ES2701188T3 - Antibody-drug conjugates and immunotoxins - Google Patents

Abstract

An antibody-drug conjugate having the formula I: A- (L-D) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: A is an antibody that selectively binds endoglin; L is a linker; p is from 1 to 10; and D is a drug comprising a cytolysin of formula IV: ** Formula ** wherein: R2 is H or C1-C4 alkyl; R6 is C1-C6 alkyl; R7 is C1-C6 alkyl, CH2OR19 or CH2OCOR20, wherein R19 is alkyl, R20 is C2-C6 alkenyl, phenyl or CH2-phenyl; R9 is C1-C6 alkyl; R10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R11 has the following structure ** Formula ** wherein R21 is H, OH, halogen, NH2, alkyloxy, phenyl, alkylamino or dialkylamino; R16 is H or a C1-C6 alkyl group; R17 is directly or indirectly linked to linker L; and q is 0, 1, 2 or 3.

Description

DESCRIPTION

Antibody-drug conjugates and immunotoxins

Field of the invention

The present invention relates to antibody-drug conjugates (CAF) and immunotoxins that target endoglin (ENG) and their use in medicine, for example, in the treatment of certain cancers.

BACKGROUND OF THE INVENTION

Malignant epithelial tumors are the leading cause of human deaths related to cancer. These solid tumors often present significant stromal reactions such as the so-called "desmoplastic stroma" or "reactive stroma", which represents 20-60% of the total tumor mass and is characterized by the existence of large amounts of stromal cells and a matrix dense extracellular (MEC). Recent studies have indicated the promoter functions of stromal cells, as exemplified by vascular cells, immune cells, fibroblasts, myofibroblasts, adipocytes, and precursor cells derived from the bone marrow ( 1-6). In particular, a considerable number of cancer-associated fibroblasts (FACs) are often observed in the tumor-associated stroma of various human cancers, including breast, lung, colon and pancreatic carcinomas ( 14, 15). Interacting in a coordinated manner with the different components of the stroma, the FACs have the ability to promote neoangiogenesis and tumor growth; It has also been shown that FACs are crucial for the development of aggressive tumors and invasion during cancer progression ( 16-25); FACs facilitate the diffusion and infiltration of tumor cells in distant organs, thus contributing to the formation of metastases. Importantly, the relevance of stromal cells has also been indicated in the failure of the systemic administration of the drug to tumors and the development of drug resistance ( 7-11). The identification of cellular and molecular targets that abolish the interactions of tumor cell-stromal cells and, therefore, attenuate tumorigenesis is currently one of the topics in translational oncology. In fact, targeting the peritumoral stroma is a fairly new strategy for treating metastatic tumors, which account for more than 90% of cancer patient mortality: only a few products have obtained therapeutic approval to date, with most they antiangiogenic drugs (Avastin®; 26). The identification and targeting of other new molecules in the tumor microenvironment is, therefore, essential to increase the effectiveness of conventional therapies in combination with therapeutic strategies based on stroma, and represents a powerful strategy for the treatment of cancer and of metastasis ( 12, 13).

Drugs based on monoclonal antibodies (MAb) represent great promise in the fight against cancer. This is because they allow the treatment to be directed at the molecular level in a precise and specific way. These advantages, together with their commercial appeal (they have short development periods, restricted competition and are easily exportable to other types of cancers once they have been approved), have pushed many pharmaceutical companies to invest in the development of new molecules based on antibodies, as well as in the licensing of new molecules or technologies of biotechnology companies.

However, despite the clinical success of therapeutic antibodies, unlabelled MAbs targeting cell surface tumor antigens rarely exhibit sufficient efficacy on their own. To increase the low activity of the MAb, the new strategies focus on joining them to toxic molecules. Plant and bacterial toxins as well as small chemotherapeutic molecules can be good candidates, since they are very potent and active in very small amounts.

The field of immunotoxins (IT) and antibody-drug conjugates (CAF) for the treatment of cancer has recently experienced a growing development activity by pharmaceutical companies, due to the technological advances made during the last years, focused on solving the problems that initially presented on immunogenicity, undesirable toxicity, production, half-life and resistance.

The immunoconjugates are made of a human recombinant antibody, humanized or chimeric, covalently bound to a cytotoxic drug. The main goal of such a structure is to unite the power of the small cytotoxic (300 to 1000 Da) and the high specificity of the MAb directed to the antigen associated to the tumor (AAT).

The Ab (of the English antibody, antibody) must be very selective to reach the antigen, whose expression must be restricted in normal cells. Ab must also be internalized efficiently in cancer cells.

The cytotoxic agent selected as the effector moiety should remove the cells only after internalization and be released into the cell cytoplasm. The payloads most commonly used in CAFs are DNA-damaging drugs such as calicheamicins, duocarmycins or compounds that target microtubules such as auristatins and matansinoids.

The Ab-cytotoxic linkers are designed to be systemically stable and to release the cytotoxic in the target cells.

AATs are usually cell membrane proteins that are overexpressed in diseased tissues or that are at least expressed sufficiently to facilitate cytotoxicity activated by internalization. Ideally, the antigen exhibits restricted expression in normal tissues with low or absent expression in the vital organs. In addition, the tumor antigen must be recognized selectively and with high affinity for an Ab.

Recent studies suggest that therapeutic agents designed to inhibit the signaling pathway of TGF-p at the tumor-stroma interface could prevent cancer progression, improving prognosis and treatment. The family of the TGF-p co-receptor is emerging as a target for cancer treatments acting on the tumor or on its neovasculature. The endoglin (ENG, CD105), an accessory protein of the TGF-p type II receptor complex, is part of this family and has the following characteristics:

Type I homodimeric membrane protein

Protein associated with cell surface angiogenesis and neovascularization in many types of cancers Overexpressed in the cells of the tumor microvasculature, specifically in angiogenic areas of the tumor No endothelium expression of normal tissue

Plasma levels correlated with breast metastasis

Internalization

Optimal accessibility of the bloodstream

Anti-ENG antibodies (eg, scFv) have been documented and their application in tumor stromal targeting strategies has been described. The specific binding, cell internalization and anti-tumor effects of anti-ENG immunoliposomes loaded with doxorubicin have been documented in vitro, using ENG + cells, and in vivo in mice. The ENG-directed conjugates of anti-EMG and ricin or natural nigrin b antibodies have been evaluated in tumor models of mice ( 27-36).

WO 2012/135517 A2 describes methods for the preparation of antibody-drug conjugates.

Ferreras et al., Toxins, 2011, Vol.3, pp. 420-441, describe the use of Sambucus ribosome inactivation proteins in the construction of immunotoxins and other conjugates for cancer therapy.

Challner, "Site-Specific Conjugation ...", PharmTech 13 Nov. 213 (internet citation) describes site-specific conjugation technologies.

WO 2008/138561 A1 describes "tubulisin derivatives", which are cytotoxic molecules and their use for the treatment of cancer and other disorders.

Van Gorp et al., Eur, J. Biochem., Vol.237, pp. 505-513, describe the characterization and molecular cloning of nigrin b, a GalNAc-specific type 2 ribosome inactivation protein.

WO 2010/126551 A1 describes the modification of linkers that bind drugs to cell binding agents, to hydrophilic linkers, resulting in improved potency in several types of cancer cells.

Gilabert-Oriol et al., Current Pharmaceutical Design, 2014, Vol. 20, No. 42, pp. 6584-6643, describes immunotoxins constructed with ribosome inactivation proteins, and the improvement in therapeutic utility thereof by using endosomal escape enhancers.

Despite these advances, there remains an unfulfilled need for additional therapeutic strategies for the treatment of tumors, including epithelial tumors, and for components for their use in such therapeutic strategies. The present invention encompasses these and other needs.

BRIEF DESCRIPTION OF THE INVENTION

In general terms, the present invention relates to anti-ENG-drug antibody conjugates as defined in claim 1. In particular, the present inventors have discovered that anti-ENG antibodies as described herein, present a highly specific union and a fast and efficient internalization.

The cytolysin derivatives are advantageously conjugated with anti-ENG antibodies for use in the treatment of tumors.

Accordingly, in a first aspect, the present invention provides an antibody-drug conjugate having the formula I:

A- (L-D) p (I)

or a pharmaceutically acceptable salt or solvate thereof,

where:

A is an antibody that binds selectively to endoglin;

L is a linker;

p is from 1 to 10; Y

D is a drug comprising a cytolysin of formula IV:

where:

R (i) 2 * is H or C ¹ -C ⁴ alkyl;

R6 is C ¹ -C ⁶ alkyl;

R7 * is C ¹ -C ⁶ alkyl, CH ² OR ¹⁹ or CH ² OCOR ²⁰ , wherein R ¹⁹ is alkyl, R ²⁰ is C ² -C ⁶ alkenyl, phenyl or CH ² -phenyl;

R 9 is C ¹ -C ⁶ alkyl;

R 10 is H, OH, O-alkyl or O-acetyl;

f is 1 or 2;

R11 has the following structure:

where

R21 is H, OH, halogen, ^NH2, alkyloxy, phenyl, alkylamino or dialkylamino;

R16 is H or a C ¹ -C ⁶ alkyl group;

R17 is linked directly or indirectly to the linker L; Y

q is 0, 1.2 or 3.

In some cases, in accordance with this and other aspects of the present invention, A is a monoclonal antibody or a binding fragment thereof that selectively binds to an extracellular region of human endoglin. In particular cases, A can comprise the determinant regions of complementarity 1-3 of the heavy chain (CDRH1-3) and the complementarity determining regions 1-3 of the light chain (CDRL1-3) having the following amino acid sequences:

(i) CDRH1: SEQ ID NO: 7 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 7;

(ii) CDRH2: SEQ ID NO: 8 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 8;

(iii) CDRH3: SEQ ID NO: 9 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 9;

(iv) CDRL1: SEQ ID NO: 10 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 10;

(v) CDRL2: SEQ ID NO: 11 or a variant thereof having up to 1 or 2 amino acid substitutions in comparison with the sequence of SEQ ID NO: 11; Y

(vi) CDRL3: SEQ ID NO: 12 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 12.

In certain cases, CDRH1-3 comprise the amino acid sequences of SEQ ID NO: 7-9, respectively, and CDRL1-3 comprise the amino acid sequences of SEQ ID NO: 10-12, respectively.

In certain cases, A comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, 95% or 99% identity with the full length sequence of SEQ ID NO: 5.

In certain cases, A comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5.

In certain cases, A comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90%, 95% or 99% identity with the full length sequence of SEQ ID NO: 6. In particular, A may comprise a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6.

In certain cases, A comprises a heavy chain comprising an amino acid sequence having at least 90%, 95% or 99% identity with the full length sequence of SEQ ID NO: 3. In particular, A may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 3.

In certain cases, A comprises a light chain comprising an amino acid sequence having at least 90%, 95% or 99% identity with the full length sequence of SEQ ID NO: 4. In particular, A may comprise a light chain comprising the amino acid sequence of SEQ ID NO: 4.

In certain cases, A may be an anti-endoglin competitive binding antibody that is structurally different from the anti-endoglin antibody molecules exemplified herein. For example, A can be an anti-endoglin antibody molecule that competes with the human anti-endoglin IgG1 antibody, identified herein as "A5", by binding to immobilized recombinant human endoglin. A5 has the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4.

In certain cases, A is a monoclonal antibody or a binding fragment thereof that selectively binds to an extracellular region of murine endoglin. Antibody-drug conjugates targeting the murine endoglin find particular use in preclinical trials, for example, use of well-characterized murine models of various cancers. In particular, A can comprise the determinant regions of complementarity 1-3 of the heavy chain (CDRH1-3) and the complementarity determining regions 1-3 of the light chain (CDRL1-3) having the following amino acid sequences:

(i) CDRH1: SEQ ID NO: 19 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 19;

(ii) CDRH2: SEQ ID NO: 20 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 20;

(iii) CDRH3: SEQ ID NO: 21 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 21;

(iv) CDRL1: SEQ ID NO: 22 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 22;

(v) CDRL2: SEQ ID NO: 23 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 23; Y

(vi) CDRL3: SEQ ID NO: 24 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 24. For example, CDRH1-3 may comprise the sequences of amino acids of SEQ ID NO: 19-21, respectively, and CDRL1-3 can comprise the amino acid sequences of SEQ ID NO: 22-24, respectively.

In certain cases, A comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, 95% or 99% identity with the full length sequence of SEQ ID NO: 17, for example, A may comprise a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17.

In certain cases, A comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90%, 95% or 99% identity with the full length sequence of SEQ ID NO: 18, for example, A may comprise a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18.

In certain cases, A comprises a heavy chain comprising an amino acid sequence having at least 90%, 95% or 99% or even 100% identity with the full length sequence of SEQ ID NO: 15 .

In certain cases, A comprises a light chain comprising an amino acid sequence having at least 90%, 95% or 99% or even 100% identity with the full length sequence of SEQ ID NO: 16 .

In certain cases, A may be an anti-endoglin competitive binding antibody that is structurally different from the anti-endoglin antibody molecules exemplified herein. For example, A can be an anti-endoglin antibody molecule that competes with the murine anti-endoglin IgG1 antibody, identified herein as "mE12", by binding to immobilized recombinant murine endoglin. The mE12 has the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16.

In accordance with this and other aspects of the present invention, D comprises a cytolysin of formula IV:

where:

R2 H or C ¹ -C ⁴ alkyl;

R6 is C ¹ -C ⁶ alkyl;

R7 is C ¹ -C ⁶ or CH2OCOR20 CH2OR19, wherein R19 is alkyl, R20 is alkenyl ^C2 - ^C6, phenyl or CH2-phenyl; R 9 is C ¹ -C ⁶ alkyl;

R 10 is H, OH, O-alkyl or O-acetyl;

f is 1 or 2;

R11 has the following structure:

where

R21 is H, OH, halogen, NH2, alkyloxy, phenyl, alkylamino or dialkylamino;

R16 is H or a C ¹ -C ⁶ alkyl group;

R17 is linked directly or indirectly to the linker L; Y

q is 0, 1, 2 or 3.

In some cases, R17 is C (O) X, CONHNHX, OX, NHX or SX, where X is a link to the linker L.

In some cases, the linker L may additionally comprise a spacer.

In some cases, the spacer has a chain length of 2 to 30 atoms.

In some cases, the spacer comprises or consists of an alkylene (i.e., divalent alkyl) or heteroalkylene (i.e., divalent heteroalkyl) group. In some cases, the spacer comprises or consists of a group alkylene or oxyalkylene.

In some cases, the spacer comprises or consists of a group - (CH ² ) n- or - (OCH ² CH ² V, where n> 1.

In some cases, the spacer comprises or consists of a group - (OCH ² CH ² V, where n> 1. In particular, n can be 1 to 15, 1 to 10, 1 to 6, or 2 to 5. By example, n can be 3 or 4.

In some cases, the spacer comprises between one and six ethylene glycol units, for example, a triethylene glycol. In some cases, the spacer can be linked directly to the group R17, or it can be linked to the group R17 through a bridge group.

In some cases, the spacer binds to the group R17 through a bridge group -C (O) X, where X is a bond to R17.

In some cases, R17 is CONHNHX and the spacer is linked to the group R17 through a bridge group -C (O) X, where X represents the junction between the spacer and R17.

In some cases, R17 is CONHNHX and the spacer is a - (OCH ² CH ² V linked to R17 through a bridge group -C (O) X, where n = 2, 3 or 4.

In some cases, D comprises a cytolysin having the following structure:

In some cases, D comprises a cytolysin having the following structure:

In certain cases, L comprises a binding group for binding to A and a cleavable portion of protease. For example, L may comprise a valine-citrulline unit. In particular, L can comprise maleimidocaproyl-valinacitrulina-p-aminobencilcarbamate.

In some cases, the double bond of the maleimide reacts with a thiol group of a cysteine residue of antibody A to form a sulfur-carbon bond in order to effect the binding of linker L to antibody A.

In some cases, -LD has a structure selected from the group consisting of:

In certain cases, -L-D can have the following structure:

According to this and other aspects of the present invention, p may, in some cases, be in the range of 1 to 5, for example, 1 to 4 or 1 to 3. In particular cases, p may be 1 or 2. In particular cases, p may be 3 or 4.

In a second aspect of the present invention there is provided an antibody-drug conjugate as defined according to the first aspect of the invention for use in medicine.

In a third aspect, the present invention provides an antibody-drug conjugate as defined according to the first aspect of the invention for use in a method of treating a tumor in a mammalian subject. In certain cases, the antibody-drug conjugate is for use in the treatment of a blood neoplasm. In certain cases, the antibody-drug conjugate is for use in the treatment of a solid tumor. In particular, the antibody-drug conjugate may be for use in the treatment of a pancreatic cancer, Ewing's sarcoma, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer or colon cancer.

In some cases, the antibody-drug conjugate is for simultaneous, sequential or separate administration with one or more antitumor drugs. The one or more antitumor drugs comprise a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent. In some cases, the one or more antitumor drugs comprise Gemcitabine, Abraxane bevacizumab, itraconazole, carboximidotriazole, an anti-PD-1 molecule or an anti-PD-LI molecule (e.g., nivolumab or pembrolizumab).

In a fourth aspect, the present invention provides an antibody-drug conjugate of the first aspect of the invention for use in the treatment of an inflammatory condition (e.g., rheumatoid arthritis) or an ocular disease (e.g., diabetic retinopathy, macular degeneration , such as wet macular degeneration related to age).

The present invention includes the combination of the features and preferred features described, except where such a combination is clearly unacceptable or is expressly indicated to have to be avoided. These and other aspects and embodiments of the invention are described in more detail below and with reference to the examples and appended figures.

Brief description of the figures

Figure 1 shows the ELISA results of mE12-IgG for binding to recombinant mouse ENG (aa 27 581). BSA coated was included as a negative control. A concentration-dependent binding to the mouse ENG was observed;

Figure 2 shows the flow cytometric analysis of mE12-IgG binding to a) B16 cells using 50 μg / ml antibody, b) B16 cells using 5 μg / ml antibody, and c) HT1080 cells included as negative control. Shaded area: single cells, non-shaded area: cells incubated with antibody;

Figure 3 shows a) ELISA binding of A5 IgG 1 to the immobilized recombinant human ENG. b) Flow cytometric analysis of A5-IgG binding to HT1080 cells;

Figure 4 shows the MALDI-Tof profile of the recombinant nigrin b chain A. Observed mass (Da): 28546.55; Expected mass (Da): 28546.09; Deviation of the mass: 0.5; Accuracy of the dough: 16 ppm.

Figure 5 shows the activity of the ribosome inactivating protein (RIP) of the recombinant nigrin b chain (recNgA) tested in the reticulocyte cell lysate ( RRL) versus nigrin b natural (TS) (3a, 3b, 6c, 9c) represents different recNgA formulations

Figure 6 shows the cytotoxicity of recNgA tested in the HT1080-FAP cell line through the viability assay of crystal violet (natural nigrina - diamonds, recombinant nigrin b chain - squares)

Figure 7 shows the RIP activity of conjugates of recNgA in a natural nigrin b: diamonds test (♦); recNgA: squares (■); conjugates of recNgA in HPS-124-37-1 (triangles: ▲) HPS-124-37-2 (crossovers: X )

Figure 8 shows the general structure of antibody conjugate for an antibody conjugated with cytolysin via a vcPABA linker. The binding of cytolysin can be through Ri or R ⁴ (identified by arrows)

Figure 9 shows the internalization analysis of anti-huENG A5 IgG 1 by cell discrimination (n = 10-30) showing only membrane staining (PM), intracellular PM staining, or only staining intracellular

Figure 10 shows the in vitro cytotoxic effect of CAF on ( A ) wild type (TS) and ( B ) HT1080 cells expressing the antigen (AG). The arrest of cell proliferation was evidenced by crystal violet staining after a 72 hour incubation of each compound at a concentration range of 10-6 to 10-12M. TAM334 parental cytolysin was used as a positive control for nonspecific cytotoxicity.

Figure 11 shows the effect of tumor growth inhibition of immunoconjugates of recNgA and cytolysin.

(A) conjugates of recNgA, administered as a single agent (OMTX505) or in combination with gemcitabine (OMTX505: GEM); (B) conjugates TAM471 (OMTX705-471) versus TAM551 (OMTX705-551); (C) conjugates TAM471 (OMTX705-471) and TAM553 (OMTX705-553) versus TAM558 (OMTX705-558). Vehicle and GEM (Gemcitabine): negative and positive control groups.

Detailed description of the invention

In the description of the present invention, the following terms will be employed, and are intended to be defined as indicated below.

Endoglina

As used, "endoglin" can be an endoglin protein of any mammalian species. In some cases, the endoglin is the human endoglin (also known as CD105, ENG or END), whose amino acid sequence is revealed in the UniProt Registration No. P17813 (Version 154, dated November 13, 2013) (SEQ ID NO: 27). In some cases, a molecule that binds endoglin (eg, an antibody molecule or a conjugate thereof) can bind to a region of the extracellular domain of endoglin. The extracellular domain of the human endoglin comprises residues 26-561 of the full-length human endoglin protein. In some cases, the endoglin is the murine endoglin (also known as CD105, antigen MJ7 / 18, ENG or END), whose amino acid sequence is revealed in UniProt Register No. Q63961 (Version 104, dated November 13, 2013) (SEQ ID NO: 28). The extracellular domain of the murine endoglin comprises residues 27-581 of the full-length murine endoglin protein.

Conjugate

As used herein, "conjugate" includes the resulting structure formed by the binding of molecules, and specifically includes antibody-drug conjugates (CAF) and immunotoxins (IT).

It binds selectively

The terms "selectively bind" and "selective link" refer to the binding of an antibody or the binding of a fragment thereof to a predetermined molecule (e.g., an antigen) in a specific manner. For example, the antibody or the binding fragment thereof, can bind to the endoglin, for example, an extracellular portion thereof, with an affinity of at least about 1x107M-1, and can bind to the predetermined molecule with an affinity that is at least two times greater (for example, five times or ten times greater) than its affinity for binding to a molecule other than the predetermined molecule.

Antibody molecule

As used herein with respect to all aspects of the invention, the term "antibody" or "antibody molecule" includes any immunoglobulin, either natural or partially or completely synthetically produced. The term "antibody" or "antibody molecule" includes monoclonal antibodies (mAbs) and polyclonal antibodies (including polyclonal antisera). The antibodies may be intact or fragments derived from whole antibodies (see below). The antibodies can be human antibodies, humanized antibodies or antibodies of non-human origin. "Monoclonal antibodies" are homogeneous populations of highly specific antibodies directed against a single antigenic site or "determinant" of the target molecule. "Polyclonal antibodies" include heterogeneous populations of antibodies that target different antigenic determinants of the target molecule. The term "antiserum" or "antisera" refers to blood serum containing antibodies obtained from immunized animals.

It has been shown that fragments of a complete antibody can perform the function of binding antigens. Therefore, the reference to antibodies herein and with reference to the methods, kits and kits of the invention, encompasses a complete antibody and also encompasses any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are (i) the Fab fragment consisting of the domains Vl, Vh, Cl and Chl; (ii) the Fd fragment consisting of the VH and Ch1 domains; (iii) the Fv fragment consisting of the Vl and Vh domains of a single antibody; (iv) the dAb fragment consisting of a Vh domain; (v) isolated CDR regions; (vi) F (ab ') ² fragments, a bivalent fragment comprising two joined Fab fragments (vii) single chain Fv molecules (scFv), in which a Vh domain and a Vl domain are linked by a peptide linker that allows the two domains associate to form an antigen-binding site; (viii) single chain Fv dimers (WO 93/11161) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; 58). The Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bridges that bind the VH and VL domains. Minibodies comprising a scFv linked to a CH3 domain can also be prepared.

In relation to an antibody molecule, the expression "binds selectively" can be used herein to refer to the situation in which a member of a specific binding pair will not exhibit any significant binding to other molecules that do not be your specific union members. The expression can also be applied when, for example, an antigen binding site is specific for a particular epitope carried by a series of antigens, in which case, the specific binding member carrying the antigen binding site will be capable of of joining the various antigens that carry the epitope.

In some cases, according to the present invention, the antibody can be a fully human antibody.

Cytotoxic chemotherapeutic agents

In some cases, according to any aspect of the present invention, the antibody-drug conjugate of the invention can be administered with, or for administration with, (either simultaneously, sequentially or separately) other antitumor drugs, including, but without limitation, a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

Cytotoxic chemotherapeutic agents are well known in the art and include anticancer agents such as:

Alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; 10 ethylene imines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U) and streptozoein (streptozotocin); Y

triacenes such as decarbazine (DTIC;

dimethyltriazenoimidazolecarboxamide);

Antimetabolites including folic acid analogues such as methotrexate (ametopterin); pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxicoformycin). Natural products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorabicin (daunomycin, rubidomycin), doxorubicin, bleomycin, plicamycin (mitramycin) and mitomycin (mitomycin Q) enzymes such as L-asparaginase, and biological response modifiers such as interferon alfenomas. miscellaneous which include platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin, anthracenedione such as mitoxantrone and antbracicline, substituted urea such as hydroxyurea, methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH), an adrenocortical suppressor such as mitotane (o, p'-DDD) and aminoglutethimide, taxol and analogues / derivatives, and hormonal agonists / antagonists such as flutamide and tamoxifen.A preferred additional cytotoxic agent is Gemcitabine (Gemzar.RTM.) A preferred additional cytotoxic agent is Paclitaxel. bound to human serum albumin (Abraxane®).

Antiangiogenic agents are well known in the art and include anticancer agents such as bevacizumab, itraconazole and carboxyamidotriazole.

Immunotherapeutic agents are well known in the art and include, for example, anti-programmed cell death protein 1 (PD-1) antibodies and anti-programmed death-ligand 1 (PD-L1) antibodies, including Nivolumab (MDX1106) and Pembrolizumab (MK-3475).

Pharmaceutical compositions

The antibody-drug conjugates of the present invention can be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient can be a compound or a combination of compounds that enters a pharmaceutical composition that does not cause side reactions and that allows, for example, the facilitation of the administration of the antibody-drug conjugate, an increase in its duration and / or in its effectiveness in the body or an increase in its solubility in solution. These pharmaceutically acceptable carriers are well known and will be adapted by the person skilled in the art depending on the mode of administration of the antibody-drug conjugate.

In some embodiments, the antibody-drug conjugates of the present invention can be provided in a lyophilized form for reconstitution prior to administration. For example, lyophilized antibody-drug conjugates can be reconstituted in sterile water and mixed with saline prior to administration to an individual.

The antibody-drug conjugates of the present invention will generally be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the conjugate drug antibody. Therefore, the pharmaceutical compositions may comprise, in addition to the antibody-drug conjugate, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the antibody-drug conjugate. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

For intravenous administration, for example, by injection, the pharmaceutical composition comprising the antibody-drug conjugate may be in the form of a parenterally acceptable aqueous solution without pyrogen and has a suitable pH, isotonicity and stability. Those skilled in the art will be well able to prepare suitable solutions using, for example, isotonic vehicles, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3'-pentanol, and mcresol) ; low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (for example, Zn-protein complexes); and / or nonionic surfactants, such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

Subject

The subject can be a human being, a companion animal (for example, a dog or cat), a laboratory animal (for example, a mouse, a rat, a rabbit, a pig or a non-human primate), an animal domestic or farm (for example, a pig, a cow, a horse or a sheep). Preferably, the subject is a human being. In some cases, the subject may be a human being diagnosed with or classified as being at risk of developing a cancer, for example, an epithelial tumor, a solid tumor or a blood neoplasm. In certain cases, the subject may be a laboratory animal, for example, a mouse model of a cancer. In certain cases, the subject may be a mammal (e.g., a human) who has been diagnosed with or classified as being at risk of developing an inflammatory condition, such as osteoarthritis or rheumatoid arthritis (RA). In particular, the subject can be a human being who has osteoarthritis or RA. In certain cases, the subject may be a mammal (e.g., a human being) who has been diagnosed with or classified as being at risk of developing an ocular disease, such as diabetic retinopathy or macular degeneration.

Cancer

The anti-ENG-drug antibody conjugates described herein find their use in the treatment of a tumor in a mammalian subject. The tumor can be a solid tumor. In particular, the cancer can be a pancreatic cancer, breast cancer, melanoma, Ewing's sarcoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer or colon cancer.

Inflammatory condition

In some cases according to the present invention, the antibody-drug conjugate of the invention may be for use in the treatment of an inflammatory condition. ENG expression in osteoarthritis and rheumatoid arthritis has been documented. See, for example, Szekanecz, Z. et al. Clinical Immunology and Immunopathology, 1995, 76, 187-194, and Leask A et al., Arthritis & Rheumatism, 2002, 46, 1857-1865. The present inventors believe that the antibody-drug conjugates described herein are capable of improving osteoarthritis, rheumatoid arthritis and / or symptoms of osteoarthritis or rheumatoid arthritis.

Eye disease

In some cases according to the present invention, the antibody-drug conjugate of the invention may be for use in the treatment of an ocular disease (e.g., diabetic retinopathy or macular degeneration, such as age-related macular degeneration). The expression of ENG has been documented in certain ocular conditions, including macular degeneration and retinopathy. See, for example, Tsutomu Yasukawa et al., Curr Eye Res. 2000, 21, 952-961 and Abu El-Asrar AM et al., Mediators Inflamm. 2012,2012: 697489, and Malik RA et al., J Cell Mol Med. 2005, 9: 692-7. The present inventors believe that the antibody-drug conjugates described herein, are capable of improving ocular diseases and / or symptoms thereof (including diabetic retinopathy or macular degeneration, such as age-related macular degeneration). . The following is presented by way of example and should not be construed as limiting the scope of the claims.

Examples

Example 1- Production of anti-ENG antibodies

The anti-ENG scFvs from the synthetic antibody phage libraries have been described previously ( 29). A scFv, directed against the extracellular region of the human ENG, known as "A5" (29) and a scFv, directed against the extracellular region of the murine ENG, known as "mE12" ( 31) were converted to IgG Full length for subsequent characterization studies and for the generation of immunotoxins and CAF. All the scFv were produced in E. coli and purified by IMAC, the IgG were produced in mammalian cells (CHO) using the Lonza GS expression vectors pEE6.4 and pEE14.4 developed for the production of antibodies. The characteristics of the scFv starting material are summarized in Table 1.

Ta l 1: n i r ifi i l v r m m l l

All scFv were produced bacterially in E. coli TG1 and were purified from the periplasmic extracts of 1L cultures by IMAC.

The plasmids corresponding to the full-length IgG1 antibodies were generated and transfected into CHO cells for the production of antibodies in the Lonza CHO expression system with yields of approximately 1 mg / l of cell culture (at the scale of laboratory). The antibodies were purified from the cell culture supernatant by protein A chromatography. The purified proteins were characterized by SDS-PAGE and size exclusion chromatography. The bioactivity was analyzed by ELISA using the recombinant ENG and the detection of antibodies bound with anti-human IgG antibodies conjugated with HRP. Cell binding was analyzed by flow cytometry using the mouse B16 cell expressing ENG.

Results:

Generated (and sequenced) plasmids:

A5 IgG1: pEE14.4 A5-IgG1 OCMTX003p (human IgG1 anti-huENG) mE12 IgG1: pEE14.4 mE12-IgG1 OCMTOO4p (human IgG1 anti-muENG)

Example 2 - Characterization of anti-ENG antibodies

The amino acid sequences of the heavy chain (HC ) and light chain (LC ) of the human anti-ENG IgG1 A5 (A5-IgG1), respectively, are shown below:

A5-IgG1-HC anti-human endoglin:

METDTLLLWVLLLWVPGSTG

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYMSWRQAPGKGLEWSAIYGSDGPTTYADSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAVYYCARyFYTAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN

HKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

aa 44 7

PM of the HC processed 48,703

theoretical p. 8.36

Possible glycosylation site ( double underlined): N297

Mutations leading to a deficiency in CCDA and CDC are shown in bold and in italics ( see also WO 99/58572)

The sequence of the signal is shown in box

The VH domain is underlined; CDRH1-H3 are shown in bold and curved underlining.

A5-IgG1-LC anti-human endoglin:

DIELTQSPSSLSASVGDRVTITCBASOSISSSLNWYQQKPGKAPKLLIYAA.SSLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQAPMgglFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 2)

aa 214

PM of the HC processed 23,113

theoretical p.76

the sequence of the signal is boxed

the VL domain is underlined; CDRL1-L3 are shown in bold and curved underlining.

A5-IgG1-HC - without signal sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSS Y M S WRQAPGKGLEWSAIYGSDGPTTYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARyFYTAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3)

A5-IgG1-LC - without signal sequence:

DIELTQSPSSLSASVGDRVTITCBASOSISSSLNWYQQKPGKAPKLLIYAA.SSLOSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQAPMCPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 4)

A5-VH:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIYGSDGDTTYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARVFYTAGFDYWGQGTLVTVSS (SEQ ID NO: 5) A5-VL:

DIELTQSPSSLSASVGDRVTITCRASQSISSSLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQAPAKPPTFGQGTKLEIKR (SEQ ID NO: 6)

A5-CDRH1:

SYAMS (SEQ ID NO: 7)

A5-CDRH2:

AIYGSDGDTTY (SEQ ID NO: 8)

A5-CDRH3:

VFYTAGFDY (SEQ ID NO: 9)

A5-CDRL1:

RASQSISSSLN (SEQ ID NO: 10)

A5-CDRL2:

AASSLQS (SEQ ID NO: 11)

A5-CDRL3:

QQAPAKPPT (SEQ ID NO: 12)

The parameters of the complete A5-IgG are as follows:

Total length of full-length IgG ( aa): 1,322

Calculated molecular mass of full-length IgG: 143,577

Estimated extinction coefficient of full-length IgG: 195,440

0.1% Abs ( = 1 g / L) 1,361

theoretical pl: 8.36

possible glycosylation site: N297

mE12-IgG1-HC anti-murine endoglin:

METDTLLLWVLLLWVPGSTG EVQLVESGGGWQPGRSLRLSCAASGFTFSSXg ^ WRQAPGK GL VWSRINSDGSSTSYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARATGTWyMSWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYMSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

aa 446

PM of the processed HC 48,574

calculated pI 8.88

Possible glycosylation site ( double underlined): N297

Mutations that lead to a deficiency in CCDA and CDC are shown in bold and italics

The sequence of the signal is shown in box

The VH domain is underlined

mE12-IgG1-LC anti-murine endoglin:

METDTLLLWVLLLWVPGSTG

SSELIQ DPAVSVALGQTVRIT CQGDSLRSYYASWYQQKPGQAPVLVIYGK ^ RPSGIPDRFSGSSSGN

TASLTITGAQAEDEADYYCNSRDSSGTVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLI

SDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK

TVAPTECS (SEQ ID NO: 14)

aa 212

PM ( processed) 22,516

calculated pI 6.69

The sequence of the signal is shown in box

the VL domain is underlined

mE12-IgG1-HC - without signal sequence:

EVQLVESGGGWQPGRSLRLSCAASGFTFSSX g ^ WRQAPGKGLVWSRINSDGSSTSYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARATGTWyMSWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYKSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15)

mE12-IgG1-LC - without signal sequence:

SSELIQ DPAVSVALGQTVRIT CQGDSLRSYYASWYQQKPGQAPVLVIYGK ^ RPSGIPDRFSGSSSGN TASLTITGAQAEDEADYYCNSRDSSGTVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS (SEQ ID NO: 16)

mE12-VH:

EVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLVWVSRINSDGSSTSYADSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAVYYCARATGTWVMSWGQGTLVTVSS (SEQ ID NO: 17)

mE12-VL:

SSELIQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGN

TASLTITGAQAEDEADYYCNSRDSSGTVFGGGTKLTVLG (SEQ ID NO: 18)

mE12-CDRH1:

SYGMH (SEQ ID NO: 19)

mE12-CDRH2:

RINSDGSSTSYADSVKG (SEQ ID NO: 20)

mE12-CDRH3:

ATGTWVMS (SEQ ID NO: 21)

mE12-CDRL1:

QGDSLRSYYAS (SEQ ID NO: 22)

mE12-CDRL2:

GKNNRPS (SEQ ID NO: 23)

mE12-CDRL3:

NSRDSSGTV (SEQ ID NO: 24)

Purified antibodies anti-muENG mE12 and anti-huENG A5 were analyzed in ELISA for binding to recombinant ENG and using flow cytometry analysis (FACS). The affinity results for the anti-muENG IgG1 mE12 (Figures 1 and 2) and anti-huENG A5 (Figure 3) are shown, and are summarized in Table 2. Figure 1 shows the concentration-dependent binding of mE12- IgG to the recombinant mouse ENG (aa 27-581), but not to the negative control (BSA). Figure 2 shows the flow cytometry analysis of mE12-IgG binding to a) B16 cells using 50 pg / ml antibody, b) B16 cells using 5 pg / ml antibody and c) HT1080 cells included as a negative control. Figure 3 demonstrates the concentration-dependent binding of A5-IgG to human ENG by a) ELISA and b) flow cytometric analysis.

For amplification, the antibody constructs were cloned in double GS vectors (pEE14.4). The plasmids of DNA were transformed, amplified and transiently transfected in CHOK1SV cells for the evaluation of expression at a volume of 200 ml. In a second stage, the antibodies were transiently expressed in large-scale cultures of 5-10 l. The clarified culture supernatant was purified using the one-step protein A chromatography. The analysis of product quality by SE-HPLC, SDS-PAGE and LAL was carried out using purified material at a concentration of 1 mg / ml, together with an internal human antibody as a control sample.

The purified protein samples were filtered through a 0.2 μm filter and analyzed by SE-HPLC chromatograms. The mE12-IgG was purified at> 98.8%. The A5-IgG was purified at> 90%. The endotoxin levels were <0.5 EU / mg.

All purified proteins were analyzed by SDS-PAGE under reducing and nonreducing conditions (data not shown).

The purified proteins A5-IgG and mE12-IgG were characterized by SDS-PAGE and size exclusion chromatography. The bioactivity was analyzed by ELISA, using the mouse / human recombinant ENG and the detection of antibodies bound with anti-human IgG antibodies conjugated with HRP. Cell binding was analyzed by flow cytometry, using HT1080 positive in ENG and mouse eEnd2 expressing ENG. The melting points were determined by dynamic light scattering using a nano zetasizer. The affinities were determined by QCM using an Attana A100. The internalization study was performed by indirect immunofluorescence confocal microscopy in permeabilized cells, detecting antibodies bound and internalized with a secondary antibody labeled with FITC.

The full-length IgG 1 purified antibodies were successfully produced both on a laboratory scale and on a large scale, for the generation of immunoconjugates. A summary of the properties of the antibody is shown in Table 2. The antibodies retained their specificity, as shown by ELISA and flow cytometry experiments. The affinities, as determined by QCM, were comparable with those of the parental antibodies. The QCM measurements indicated the contribution of the avidity effects to the high affinity binding. The thermal stability differed between the different IgG (64-72 ° C).

Preliminary internalization studies indicate rapid cellular internalization for A5-IgG to cells expressing human ENG. In fact, in 30 minutes, almost 90% of A5-IgG is found in HT1080 cells.

T l 2: R m n l r i l n i r

Figure 9 shows the internalization of anti-huENG IgG 1 A5.

Comparative Example 3 - A chain of Nigrina b

In order to avoid the side effects of the free toxin that could be released into the bloodstream and to reduce the possible immunogenicity of the RIP toxin, as widely described with ricin, the enzymatic domain of nigrin b, the chain A, was cloned and expressed in the bacteria. The present inventors hypothesized that, if the A chain produced in the bacteria was able to retain its activity, it would not be able to enter the cells, unless it is conjugated with a vehicle molecule, such as an antibody.

Production

The A chain of nigrin b was synthesized taking into account codon optimization for bacterial expression and the synthesized gene was cloned into two different vectors, Nigrin_pET30b-3 and Nigrin_pET33b-1 (His / - labeling) for expression in two different strains of E. coli, E. coli BLR (DE3) and E. coli HMS174 (DE3). Different culture media were used to check the different expression conditions. The purification process was established using Capto Q chromatography and High Performance Sepharose SP. The A chain of purified recombinant nigrin b (recNgA) was formulated at 5 mg / ml in 1X PBS at pH 7.4, 0.5 mM DTT, 10% glycerol. The endotoxin levels were <1 EU / mg of nigrin and the purity was> 99% in monomeric form.

The N-terminal sequencing of Eldman revealed that the N-terminal end of recNgA corresponded to the expected sequence.

A chain amino acid sequence of recombinant nigrin b:

MIDYPSVSFNLDGAKSATYRDFLSNLRKTVATGTYEVNGLPVLRRESEVQVKSRFVLVPLTNYNGNTV

TLAVDVTNLYWAFSGNANSYFFKDATEVQKSNLFVGTKQNTLSFTGNYDNLETAANTRRESIELGPS

PLDGAITSLYHGDSVARSLLWIQMVSEAARFRYIEQEVRRSLQQATSFTPNALMLSMENNWSSMSLE

IQQAGNNVSPFFGTVQLLNYDHTHRLVDNFEELYKITGIAILLFRCSSPSND (SEQ ID NO: 25)

The recombinant nigrin b chain A has the following characteristics:

Number of amino acids: 256

Molecular weight: 28546.0

theoretical p.:5.45

The nucleotide sequence encoding nigrin b chain A is as follows:

atagactatc cctccgtctc cttcaacttg gatggagcca agtcggctac atacagggac ttcctcagca aacagtggca acctgcgaaa actggcacct atgaagtaaa cggtttacca gtactgaggc gcgaaagtga agtacaggtc aagagtcggt tcgttctcgt ccctctcacc aattacaatg gaaacaccgt cacgttggca gtagatgtga ccaaccttta cgtggtggct tttagtggaa atgcaaactc ctactttttc aaggacgcta cggaagttca aaagagtaat ttattcgttg gcaccaagca aaatacgtta tccttcacgg gtaattatga caaccttgag actgcggcga atactaggag ggagtctatc gaactgggac ccagtccgct agatggagcc attacaagtt tgtatcatgg tgatagcgta gcccgatctc tccttgtggt aattcagatg gtctcggaag cggcaaggtt cagatacatt gagcaagaag tgcgccgaag cctacagcag gctacaagct tcacaccaaa tgctttgatg ctgagcatgg agaacaactg gtcgtctatg tccttggaga tccagcaggc gggaaataat gtatcaccct tctttgggac cgttcagctt ctaaattacg atcacactca ccgcctagtt gacaactttg aggaactcta taagattacg gggatagcaa ttcttctctt ccgttgctcc tcaccaagca atgat (SEQ ID NO: 26)

materials

- Genetic construction of Nigrina pET30b-3.

- Escherichia coli (Migula) Castellani and Palmers BLR (DE3)

- Culture media: self-induced medium (AIM )

- Extraction culture buffer: 10 mM glycine / NaOH, 1 μg / ml Leupeptin, 1 μg / ml Pepstatin, pH 9.5.

- Extraction supernatant buffer 50 mM Tris-HCl, 200 mM NaCl, 2 mM MgCl 2, 1 jgm l-1 leupeptin, 1 jgm l-1 pepstatin, 0.1 mg ml-1 lysozyme, pH 8.0.

- Dialysis solution: Citric acid / 25 mM NaOH at pH 5.0. -CAPT Q FPLC: Balancing buffer A: 50 mM glycine / NaOH at pH 9.5. Elution Buffer B: 50 mM Glycine / NaOH at pH 9.5, 1 M NaCl

- Grouped fractions of the Capto Q stage (+ 80 ml extraction).

- SP Sepharose HP FPLC: Balancing buffer A: 25 mM citric acid at pH 4.0. Elution Buffer B: Citric Acid 25 mM at pH 4.0, 1 M NaCl.

Methods

E. coli BLR (DE3) that maintains the expression of Nigrina pET30b-3 cultivated in 1 liter of self-inducible medium format (AIM) with 30 jg m l-1 of Kanamycin. The expression of the protein was triggered by lactose activation and glucose depletion after approximately 3-4 hours of growth. Afterwards, the temperature was reduced to 20 ° C for an overnight duration.

For extraction, each cell pellet was initially resuspended in 80 ml of extraction buffer per liter of culture, and 3 disintegration cycles of 7 minutes were performed at 1100-110 Bar after 30 minutes of incubation at 8 ° C under agitation. The extract was then subjected to 60 minutes of centrifugation at 15,900 g, at 8 ° C. The supernatant was the starting material of the purification.

Capto Q FPLC: 160 ml of product extracted from culture 81 were loaded into 160 ml of Capto Q and equilibrated using 4CV of equilibration buffer and washed with 15 CV of equilibration buffer. The elution was carried out in three stages: 15CV at 1.5mS / cm (7.6% B); 20 CV at 23.8 mS / cm (18.9% B); 20CV 100% B.

The dialysis was carried out under the following conditions: 650 ml of the product were subjected to dialysis in 4x5l baths in citric acid / 25 mM NaOH at pH 5.0, cut-off value 6-8000Da. Dialysis factor ~ 3500, <24 h. After dialysis, a centrifugation of 30 minutes at 20,500 g and at 8 ° C allowed to separate the soluble fraction from the insoluble fraction. SDS-PAGE was performed on total and soluble fractions both before and after dialysis (10 μl loaded on SDS-PAGE). The eluent was dialyzed in PBS at pH 7.4 and filtered at 9 = 0.22 jm using filters of 2x20cm2 EKV.

SP Sepharose HP: 610 ml of dialysed mixture of Capto Q in 25 mM citric acid at pH 5.0 in 240 ml of high-performance SP Sepharose with 4 CV of equilibration buffer was charged and washed with 15 CV of equilibration buffer and eluted at a gradient of 25 CV to 20% B; 4CV stage of 100% of B.

The mixed fractions from the HP SP Sepharose step were dialyzed in PBS at pH 7.4, 0.5 mM DTT (5 x 4 baths, pooled fractions from 950 ml to 0.97 mg / ml). The cut-off value was 6-8000 Da, the dialysis factor was ~ 3130, time> 24 h. Then, a 30 min centrifugation at 20.55 g and 8 ° C allowed the soluble fraction to be separated from the insoluble fraction. Then 10% glycerol was added.

Finally, the eluent was submitted to dialysis in PBS at pH 7.4 (5 baths ~ 3100) and filtered at 9 = 0.2 jm, then the batch recNg bA was frozen instantaneously at -80 ° C. Later, a SEC was carried out in a Semi-Preparative S200 Superdex.

Size exclusion chromatography and mass spectrometric analysis demonstrated the monomeric and purification status of the obtained recombinant nigrin b chain (recNgA) (Figure 4).

Stability studies were conducted to evaluate the effect of pH and temperature on the nigrin b chain protein A itself and its activity. The recNgA is stable at a pH that varies from 5 to 9, and in the presence or absence of glycerol (from 10 to 45%) (data not shown).

Activity

The activity of the ribosome inactivating protein (RIP ) of the n- chain A of nigrin b in lysate without cells of the rabbit reticulocytes was evaluated: the IC 50 value obtained was similar to the natural nigrin b and in a range of 2.5 to 25 pM (see Figure 5). Therefore, the A chain of nigrin b, expressed as a recombinant protein in bacteria, maintains its enzymatic activity, arguing that glycosylation is not required for the RIP activity of the n-chain b of nigrin b.

RecNgA retains its activity in lysates without rabbit reticulocyte cells if stored frozen at (-80 ° C) and below 3 freeze-thaw cycles (not shown).

The cytotoxic activity of recNgA was assayed in cell cultures through a viability assay based on crystal violet. recNgA, which lacks the B chain for translocation in cells, has an activity of 100 to 1000 less toxic than natural nigrin b, as shown in Figure 6. Natural nigrin b presented an IC50 == 2x10-8M (similar to the previously published data, see 37), while recNgA showed a C | 50 == 2x10-6M.

Previously published studies showed that natural nigrin b has a higher RIP activity than ricin in the r Rl assay, whereas it is much less toxic (30-10,000 times, approximately) in cells or in vivo (see the values of IC 50 and LD 50 in Table 3).

Upon removal of the B chain, the ricin A chain loses activity in both the RRL assay and the cytotoxicity assay. Unexpectedly, the A chain of nigrin b, generated for the first time in this present invention, only loses activity in the cell cytotoxicity assay, while it even increased in the RRL assay with respect to natural Nigrin b. These data suggested that, in the case of ricin, the withdrawal of the B chain was affecting not only the binding and translocation of the A chain, but also its RIP activity, although this was not the case for the chain A of nigrin b, which conserves and even increases its RRL activity with respect to its natural counterpart. As a result, the A chain of nigrin b is 50 times more active than the A chain of ricin in RRL.

Accordingly, after conjugation, the conjugates, the antibody-chain A conjugates of the b-drug nigrin have a greater cytotoxic activity (IC50 in a range of pM) than the conjugates of the A chain antibody of ricin - conjugates of the drugs (nM interval) (not shown).

Table 3: In vitro and in ^{vivid activity data} for ricin and nigrin b (natural and A chain).

Lysate IC ^{50 LD50} mouse cells

HeLa rabbit (pgkg ^{- 1} )

IC ⁵⁰ (pM) (pM)

27,600.00 (20

Nigrina b 30 2300 nM; line 12,000.00

cellular dpt)

Chain A 750,000. 00

(HT1080-FAP)

of the 6, 5 ND

nigrina b 300,000. 00

(HT1080)

Ricina 100 0.67

Chain A 260,000.00 _

of ricin 500

(T lymphocytes)

(Own data of the inventors of the n-chain b chain A, see also Ferreras J.M. et al., Toxins, 3: 420, 2011, Svinth M. et al., BBRC, 249: 637, 1998)

Comparative Example 4 - Conjugation of the A chain of nigrin b with anti-ENG antibodies

For immunoconjugates containing RIP to exhibit maximum cytotoxicity, RIP must be released from the targeting vehicle in a fully active manner, which requires avoidance of steric hindrance (38). The disulfide bond is the only type of bond that fits this criterion (39, 40). This binding allows conjugation using reagents for the introduction of free sulfhydryl groups such as N-succinimidyl 3 (2-pyridyl-dithiopropionate) (SPDP) and 4-succinimidyloxycarbonyl-a-methyl-a (2-pyridyl-dithio) toluene (SMPT) ). Immunotoxins consisting of mAb covalently bound to toxins by hindered disulfide linkers, often labeled as second generation immunotoxins, are stable, long-lasting and have potent cytotoxicity to the target cells (41).

SPDP has already been used in the preparation of immunotoxins (IT) containing nigrin b (36, 42). In addition, SMPT protects the disulfide bond from attack by thiolate anions, improving the in vivo stability of the link (43, 44).

Material

- Chain A of recombinant nigrin b in PBS, at pH 7.4, with 10% glycerol, 0.5 mM DTT, 4.92 gl-1, stored at 5 ° C.

- 5,5'-dithio-bis- (2-nitrobenzoic acid)

- Desalination columns GE PD MiniTrap G-10.

- Sterile Minisart filters of 0.2 | jm and 28 mm.

- Workstation Sciclone to Lh 3000.

- Sarstedt Microtest plate with 96 wells, flat bottom, ref. No. 82.1581.

Methods

Dithiothreitol (DTT, Cleland's reagent) is a redox agent that will be used to release the thiol groups present in the protein sample. Once said groups have been released and, therefore, are available to react, 5,5'-dithio-bis- (2-nitrobenzoic acid) (Ellman's reagent) will be added. The disulfide bridge of Ellman's reagent will be cleaved and the 2 resulting molecules of thio-nitrobenzoate (TNB) will attack the protein at the sites of the thiol group. To titrate the TNBs, absorbance values will be taken at A = 412nm, a wavelength at which DTT is not absorbed, representing the concentration of the thiol groups. The ratio of this to the concentration of the protein taken from its absorbance at A = 280 will yield the number of free thiol groups per protein molecule.

The direct thiol titration was carried out as follows:

204 μl of A of recNg b was dissolved in 796 μl of 20 mM phosphate, 250 mM NaCl, 1 mM EDTA at pH 7.0 (assay buffer) (1.0033 gl -1 = final concentration). The Ellman reagent was dissolved in 0.2 M phosphate at 3 gl-1. For both buffers, monobasic and dibasic sodium phosphate was added in a mass ratio of 1.61 to 1. The pH was adjusted to room temperature and the buffers were filtered. 100 ml of Ellman's buffer and 500 ml of buffer were prepared of testing. Ellman's reagent was completely resuspended instead of weighed.

The recNgA sample was incubated in the presence of 4.8 mM DTT at room temperature for 30 min. The sample of recNgbA was then purified on the column, and aliquots of the first 10 ml of the eluent were taken (V = 0.5 ml). The A ^{280 was taken} from the aliquots and the two most concentrated were mixed. The A ²⁸⁰ was taken again. 10 μl of 3 gl -1 of DTNB was added and the A ⁴¹² was measured after 2 min (n = 1), using Ellman diluted in assay buffer in the same concentration as a blank (nb = 3). The readings belonged to the linear interval of 0.1-3 AU. Protein solutions were pipetted just below the meniscus after vortexing. 100 μl were pipetted per well. The results of this study show that the thiol group that belongs to the single cysteine residue of recNgA is free and available for the reaction, without being blocked by its tertiary structure. This will allow recNgbA to be conjugated using a linker that requires a hindered interchain disulfide bond.

It is well established that immunoconjugates containing ribosome activation proteins exhibit maximum cytotoxicity only when the toxin molecule is released from the targeting vehicle in a fully active form. Separation of the RIP molecule from the vehicle is required to avoid steric hindrance and to allow efficient translocation of the toxin to the cytoplasm (38). Currently, the disulfide bond is the only type of link that seems to fit this criterion ( 40).

The coupling of the two different protein macromolecules, which results in the formation of heterodimers, requires that each protein be modified before mixing them to react. In the case of the A chains of the type 2 RLP, the modification is limited to the reductive cleavage of the natural cysteine residue that links the active (A) and binding (B) chains of the molecule.

For IgG molecules, this is not possible, since the cysteine residues are involved in the maintenance of the tertiary and / or quaternary structure of the protein, so that it is not possible to reduce them without loss of the specific functions of the protein. On the other hand, presumably, some of the cysteine residues are not sterically accessible, as was demonstrated by the thiol groups for immunoglobulin that had to be generated for optimal conjugation with an activated RIP ( 45).

For these reasons, in most IgG molecules, thiol groups are chemically inserted using heterobifunctional reagents, and several methods have been developed in order to generate heteroconjugates that prevent or minimize the formation of homopolymers. In most cases, the reagents used to introduce thiol groups react with the amino groups, forming amide or amidine bonds. Amino groups are reactive, abundant and, in a limited way for most proteins, replaceable. That is, a limited number of amino groups can be modified without reducing the biological activity of the protein (40).

The most commonly used reagents for the introduction of free sulfhydryl groups are N-succinimidyl 3 (2-pyridyldithiopropionate) (SPDP) and 4-succinimidyloxycarbonyl-a-methyl-a (2-pyridyl-dithio) toluene (SMPT), which introduce groups 2-pyridyl disulfide in the protein by reacting them with the amino groups to form neutral amides and methyl 4-mercaptobutyrimidate (2-iminothiolane, Reagent from Traut) that introduces mercaptobutyrimidoyl groups, which react to form charged amidines, thus preserving the positive charge of the derivatized amino acid (40; 44).

SPDP and SMPT introduce a hindered disulfide bond, while the -SH of 2-iminothiolane must be protected by reacting it with 5,5'-dithiobis-2-nitrobenzoic acid (Ellman's reagent).

The reaction with the Ellman reagent is also used to rapidly measure the sulfhydryl groups of the protein (45, 46).

SMPT has a methyl group and a benzene ring attached to the carbon atom adjacent to the disulfide bond that protects it from attack by thiolate anions, thereby improving the in vivo stability of the link (43, 44).

According to these data, the IgG proteins can be modified with SMPT, which does not significantly affect the antigen-binding property of the molecules under the following conditions, even if they change the charge of the protein at the reaction site.

In one study, the present inventors investigated the conjugation of IgG1 with recNgA, using 2 molar proportions of recNgA: mAb other than 2.5 and 3.5, after derivatization, using a molar ratio of SMPT: mAb of 6, after the conjugation protocols (see 40). The purification was carried out by size exclusion chromatography on Sephacryl S200 (see 41).

Under the conditions described, the immunotoxin is predominantly a mixture of antibody bound to one or two molecules of toxin, with the presence of high molecular weight components (IgG linked to several RIP proteins), as well as free RIPs and polymeric (dimeric in the case of recNgA) and free antibodies. Therefore, it is thought that careful purification is desirable to obtain a pure product.

In vitro activity assay

The activity assay on conjugates prepared as described above was performed through the evaluation of RIP activity in the rabbit reticulocyte cell lysate (RRL) assay. The results are presented in Figure 7.

The IC ⁵⁰ values obtained for the nigrin or the recNgA were in the range of 2.5 pM and those for the conjugates were similar and in the range of 1-0.5 pM, even higher than the positive control of nigrin b natural, demonstrating that the antibody conjugation did not affect the enzymatic activity of recNgA.

Example 5 - Conjugation of cytolysins with anti-ENG antibodies

The cytolysins used for the conjugation studies were chosen from the general structure shown above (formula IV). These structures have activity against the different cancer cell lines (nM to pM interval).

Various linker systems can be used and linked to either the R2 or the R17 position of the molecule.

The general scheme of the cytolysin-drug antibody conjugates, including the vcPABA linker and the anti-ENG antibody, is shown in Figure 8 (in the structure depicted in Figure 8, the binding site of the cytolysin to the linker vcPABA is in position Ri or R ⁴ - the numbering system of Ri and R ⁴ used in Figure 11 differs from the numbering system of group R used, for example, in the claims, it is intended that Ri of Figure 11 corresponds to R2 in the claims and that R ⁴ of Figure 11 corresponds to R17 of the claims).

The cleavable protease linker vcPABA (valine-citrulline-PABC) has previously been used in the CAF molecule Brentuximab Vedotine, developed by Seattle Genetics and Takeda, and recently approved by the FDA and EMEA as Adcetris® (2011, and Nov. 2012). , respectively). In this CAF, vcPABA has been coupled in its free NH2 to maleimide caproil for thiol-based conjugation to mAb (anti-CD30 antibody cAC10). On the other hand, vcPABA has been conjugated through its COOH with the cytotoxic drug Auristatin from Seattle Genetics (MMAE). (see 48) The present inventors have used this linker (maleimide caproil-vcPABA) to conjugate anti-ENG antibodies through the thiol-based reaction with maleimide caproil and, at the other extreme, cytotoxic cytolysin molecules through its cyclic piperidine with vcPABA (Ri or R ^{4 positions} of the cytolysin shown in Figure 8). Synthesis of Maleimido-val-cit-PABOCO-Tubulisina / Citolisina-TAM461:

TAM461 (Tubulysin / Cytolysin) : 30.0 mg (0.041 mmol)

DMF: 3 ml

TAM465 (Linker): 35 mg (0.045 mmol)

HOBt: 1.4 mg

DIPEA: 10 Ml

TAM461 and TAM465 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18 h. The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 2-6% methanol: DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m / z = 1371 [M + H].

Synthesis of Maleimido-val-cit-PABOCO-Tubulisina / Citolisina-TAM470:

TAM470 (Tubulysin / Cytolysin) : 0.07 mmol

DMF: 5 ml

TAM466 (Linker): 50 mg (0.065 mmol)

HOBt: 2.4 mg

DIPEA: 18 Ml

TAM470 and TAM466 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18 h and then analyzed with t Lc, which indicates the completion of the reaction, The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 4-12% methanol: DCM for give 56 mg of TAM471 (yield: 62%). ESI-MS: 1384.6 [M + 1].

An assay of in vitro activity is performed . The functional activity is evaluated through the microtubule inhibition assay, while the cytotoxic activity is determined through the crystal violet viability assay.

Generation of cytolysin-linker derivatives

Different cytolysin-linker derivatives were synthesized according to the general structure presented in Figure 11, wherein the vcPABA linker was added either in the R1 (TAM467, TAM551) or R4 (TAM471, TAM553, TAM558) position, alone or with ethylene glycol spacer (EG, n = 1 to 3), or substituted by ethylene glycol groups (n = 3) (TAM552). The respective chemical structures are presented in Table 4.

Table 4: Umymic structure of cytolysin-linker derivatives

The microtubule inhibition activity and the cytotoxic activity of each new derivative were evaluated through the tubulin polymerization inhibition assay (TPI, tubulin polymerization assay kit, cytoskeleton, Cat # BK011P), and the arrest of cell proliferation in HT1080 cells (CPA, crystal violet). The IC50 was calculated and the results are presented in Table 5.

Table 5: Microtubule inhibition activity and cellular cytotoxicity activity of i li in-nl z r. ND: N rmin ^

The in vitro activity of parenteral cytolysin TAM334 is within the same range of other charges currently used for the generation of antibody-drug conjugates such as auristatins (MMAE) or maytansinoids (DM1-DM4). As expected and previously described for other compounds of the Tubulisin A family, after the addition of the linker, the cytotoxic cellular activity of the cytolysins was reduced with respect to the parental compound TAM334. In addition, the derivative of TAM467 was showing significantly lower activity in both trials. All derivatives were used in conjugation to generate CAF molecules and were evaluated comparatively both in vitro and in vivo to select the most active cytolysin-linker derivative.

Conjugation and chemical characterization of the CAF

Each of the newly generated derivatives was conjugated with monoclonal human IgG1 antibodies following a non-site specific conjugation method on cysteine residues. For this purpose, a batch of antibodies was reduced and reacted with each of the derivatives. Different proportions of TCEP were tested to achieve the optimal DAR of 3-4, less than 10% of free antibody and drug. The optimal conjugation conditions were as follows: TCEP = 2.5 and 3.57 of Ellmann's thiol levels. The antibody-drug conjugates were then purified on G25 Sephadex and analyzed by size exclusion chromatography (SEC) to determine their purity, as well as hydrophobic interaction chromatography (HIC) and reverse phase chromatography of polymeric liquid (PLRP). to determine the ARD, the content of free antibody and the distribution profile of different CAF species (0-8 drugs / mAb). The content of free drug was evaluated by the UV detection method at 280 nm. The results of the chemical analysis were determined (not shown) and the characteristics of the CAFs are shown in Table 6.

Table 6: Summary of the physical characteristics of the different CAF molecules

The different drugs produced different levels of aggregation. Specifically, CAF HPS157-039-002 (TAM551) showed the highest levels of aggregation already at DAR = 3.08, leaving 22.4% of antibody unconjugated. A preliminary conjugation with TAM467 also showed a high level of aggregation: at DAR 3.27, the purity of SEC was already only 67% with 16 % of the free drug (data not shown). These data were suggesting that the vcPABA linker at the R1 position was not optimal for this type of cytolysin molecules.

In vitro evaluation of cytolysin-drug antibody conjugates

The CAF molecules of cytolysin were evaluated comparatively in vitro by the proliferation arrest assay (crystal violet staining). The results are presented in Figure 10 and the IC 50 values in Table 7.

Table 7: Value I ni n l n n i n l r liferation (nM)

The location of the vcPABA linker only in the R1 position (CAF-551) generated antibody-drug conjugates with much less cytotoxic activity in vitro with respect to the R4 position (CAF-471) (Figure 10, Table 7). In addition, the increase in the number of ethylene glycol groups as a spacer for the vcPABA linker at the R4 position (CAF-471 (n = 0) versus CAF-553 (n = 1) and CAF-558 (n = 3)) demonstrated which increases the antigen-specific cytotoxic activity in vitro (Figure 10). In fact, while CAF-471 and c AF-553 showed low cytotoxic and non-specific antigen activity (IC ^{50 range} of 10 nM and 100 nM, respectively) without differences between the cells HT1080 of wild type (TS) and those that present the antigen (AG), CAF-558 presented a specific cytotoxic activity of interval of 1 nM with a proportion of 500 between HT1080 AG and TS cells.

Example 6 - In vivo evaluation of the antitumor effect of antibody-drug conjugates

Both types of immunoconjugates, conjugates of recNgA- and cytolysin-antibody, were evaluated for their antitumor effect in vivo in a mouse model of patient-derived xenograft for pancreatic cancer (PAXF-736) previously selected for antigen expression.

Dose interval studies were performed to define the maximum tolerated dose for use in efficacy studies (not shown). For the recNgA immunoconjugates, a higher tolerated dose of 0.5 mg / kg was found, while the lysine antibody conjugates, regardless of the derivative used, were administered at doses of 2.5 mg / kg to 20 mg / kg. kg, without any weight loss or toxic effect.

The immunoconjugates were then administered once a week intraperitoneally for 5 weeks. Tumor volume and body weight were measured every 2-3 days. PDX mice treated with vehicle and treated with Gemcitabine (150 mg / kg) were used as negative control and positive control groups, respectively. The results are shown in figure 11.

The recNgA immunoconjugates (OMTX505) exhibited high antitumor efficacy in vivo (60%) at a dose of 0.5 mg / kg in murine PDX models of pancreatic cancer (Figure 11A). When combined with gemcitabine, it even showed 100% inhibition of tumor growth and tumor regression.

According to the in vitro results (see Figure 10 and Table 7), the location of the vcPABA linker alone in the R1 position (OMTX705-551) generated antibody-drug conjugates without antitumor activity in vivo (Figure 11B). Supporting the in vitro data , the increase in the number of ethylene glycol groups as a spacer for the vcPABA linker at the R4 position (OMTX705-471 (n = 0) versus OMTX705-553 (n = l) and OMTX705-558 (n = 3)) was shown to increase the antitumor effect in vivo (Figure 11C). OMTX705-471 and OMTX705-553 showed no antitumor effect in vivo, while OMTX705-558 exhibited a tumor growth inhibition effect of 40% at a dose of 2.5 mg / kg in a mouse model of PDX for cancer of pancreas.

From these data, the recNgA and TAM558 molecules were selected as the best fillers for the anti-ENG-drug antibody conjugates.

Example 7 - Models of Ewing sarcoma

The plasticity of tumor cells allows certain types of highly malignant tumor cells to dedifferentiate and become involved in an embryonic-type multipotent plastic phenotype, which allows them to 'adapt' during tumor progression and escape from conventional therapeutic strategies. A recent study showed that the expression of ENG correlates with the plasticity of tumor cells in Ewing's sarcoma, and that it is significantly associated with a worse survival of patients with Ewing's sarcoma. Ewing's sarcoma with reduced levels of ENG showed reduced tumor growth in vivo. This study, therefore, delineates an important role of ENG in the plasticity of tumor cells and the progression of aggressive tumors ( 51).

The present inventors hypothesize the therapeutic potential of anti-ENG, IT and FAC monoclonal antibodies in the treatment of Ewing's sarcoma.

14 Ewing sarcoma cell line models have been developed for in vitro studies , and their corresponding xenograft models, for the systematic selection and characterization of therapeutic molecules for the treatment of Ewing's sarcoma. ENG expression has been confirmed in the 14 cell lines, and all samples from human patients with Ewing's sarcoma (n = 10) have been examined.

The specific embodiments described herein are offered by way of example, not by way of limitation. All subheadings in this document are included for convenience only, and should not be construed as limiting the disclosure in any way.

References

1. Weinberg, R.A., et al., Garland science, Taylor & Francis Group LLC, New York, NY, USA, 2007

2. Nieman, K.M., et al., Nat. Med., 2011, 17: 1498-1503

3. Joyce, J.A., et al., Nat. Rev. Cancer, 2009, 9: 239-252

4. Hanahan, D., et al., Cancer Cell, 2012, 21: 309-322

5. Gupta, G.P., et al., Cell, 2006: 127: 679-695

6. Valastyan, S., et al., Cell, 2011, 147: 275-292

7. Meads, M.B, et al., Nat. Rev. Cancer, 2009, 9: 665-674

Claims (23)

1. An antibody-drug conjugate having the formula I: A- (L-D) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: A is an antibody that binds selectively to endoglin; L is a linker; p is from 1 to 10; Y D is a drug comprising a cytolysin of formula IV:

where: R (i) 2 * is H or C ¹ -C ⁴ alkyl; R6 is C ¹ -C ⁶ alkyl; R7 * is C ¹ -C ⁶ alkyl, CH ² OR ¹⁹ or CH ² OCOR ²⁰ , wherein R ¹⁹ is alkyl, R ²⁰ is C ² -C ⁶ alkenyl, phenyl or CH ² -phenyl; R 9 is C ¹ -C ⁶ alkyl; R 10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R11 has the following structure:

where R21 is H, OH, halogen, ^NH2, alkyloxy, phenyl, alkylamino or dialkylamino; R16 is H or a C ¹ -C ⁶ alkyl group; R17 is linked directly or indirectly to the linker L; Y q is 0, 1, 2 or 3. 2. The antibody-drug conjugate of claim 1, wherein A comprises the determinant regions of complementarity 1-3 of the heavy chain (CDRH1-3) and the complementarity determining regions 1-3 of the light chain (CDRL1-3) ) that have the following amino acid sequences: (i) CDRH1: SEQ ID NO: 7 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 7; (ii) CDRH2: SEQ ID NO: 8 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 8; (iii) CDRH3: SEQ ID NO: 9 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 9; (iv) CDRL1: SEQ ID NO: 10 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 10; (v) CDRL2: SEQ ID NO: 11 or a variant thereof having up to 1 or 2 amino acid substitutions in comparison with the sequence of SEQ ID NO: 11; Y (vi) CDRL3: SEQ ID NO: 12 or a variant thereof having up to 1 or 2 amino acid substitutions compared to the sequence of SEQ ID NO: 12. 3. The antibody-drug conjugate of claim 2, wherein A comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6. 4. The antibody-drug conjugate of claim 2, wherein A comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4. 5. The antibody-drug conjugate of any one of the preceding claims, wherein R17 is C (O) X, CONHNHX, OX, NHX or Sx, wherein X is a linker to the linker L. 6. The antibody-drug conjugate of any one of the preceding claims, wherein the linker L further comprises a spacer. 7. The antibody-drug conjugate of claim 6, wherein the spacer has a chain length of 2 to 30 atoms. 8. The antibody-drug conjugate of claim 6 or claim 7, wherein the spacer comprises or consists of a group - (CH ² ) n- or - (OCH ² CH ² V, wherein n = 1 to 10 . 9. The antibody-drug conjugate of any one of claims 6 to 8, wherein the spacer is directly linked to the group R17, or is linked to the group R17 through a bridging group. 10. The antibody-drug conjugate of claim 9, wherein the spacer is linked to the group R17 through a bridging group -C (O) X, wherein X is a bond to R17. 11. The antibody-drug conjugate of claim 1, wherein D comprises a cytolysin having the following structure:

12. The antibody-drug conjugate of any one of the preceding claims, wherein L comprises a binding group for attachment to A and a cleavable portion of protease. The antibody-drug conjugate of claim 12, wherein L comprises maleimidocaproyl-valine-citrulline-aminobenzylcarbamate. 14. The antibody-drug conjugate of any one of the preceding claims, wherein -L-D has a structure selected from the group consisting of:

15. An antibody-drug conjugate as defined in any one of the preceding claims for use in medicine. 16. An antibody-drug conjugate as defined in any one of claims 1 to 14 for use in a method of treating a tumor in a mammalian subject. 17. The antibody-drug conjugate for use according to claim 16, wherein said antibody-drug conjugate is for simultaneous, sequential or separate administration with one or more other antitumor drugs. 18. The antibody-drug conjugate for use according to claim 17, wherein said one or more other antitumor drugs comprises a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent. 19. The antibody-drug conjugate for use according to claim 18, wherein said one or more other antitumor drugs comprise Gemcitabine, Abraxane bevacizumab, itraconazole, carboximidotriazole, an anti-PD-1 molecule or an anti-PD-molecule. LI. The antibody-drug conjugate for use according to claim 19, wherein said anti-PD-1 molecule or an anti-PD-LI molecule comprises nivolumab or pembrolizumab. 21. The antibody-drug conjugate for use according to any one of claims 16 to 20, wherein the tumor is a blood neoplasm, pancreatic cancer, Ewing sarcoma, breast cancer, melanoma, lung cancer, cancer. of head and neck, ovarian cancer, bladder cancer or colon cancer. 22. The antibody-drug conjugate of any one of claims 1 to 14 for use in a method of treating an inflammatory condition or ocular disease. 23. The antibody-drug conjugate for use according to claim 22, wherein: said inflammatory condition is rheumatoid arthritis; I said ocular disease is diabetic retinopathy or macular degeneration.